A highly specialized transport system has been characterized as an essential element of cholinergic neurons. This transport system is know as the high affinity choline uptake systems (HAChUS) and has been shown to co-transport sodium. Within the cholinergic nerve terminal acetylcholine (ACh) synthesis is tightly coupled to the activity of the choline co-transporter. Moreover, the activity of the choline co- transporter appears coupled to the activity of the cholinergic neuron. The specific cellular mechanism responsible for the coupling of neuronal activity with choline cotransporter activity is unknown. The hypothesis under test in this subproject is that the activity of the choline cotransporter is regulated via the transfer of packages of functional transporters between an intracellular compartment and the plasma membrane. Furthermore, it is hypothesized that this transfer between compartments is mediated by stimulus dependent intracellular second messengers. These hypotheses will be validated by accomplishing the following set of Specific Aims: 1. To demonstrate and intracellular or microsomal (i.e. vesicular) source of choline co-transporters and the relationship of intracellular transporters to the regulation of transporters in the plasma membrane. 2. To demonstrate the translocation of a pool of choline co- transporters from an intercellular source to the plasma membrane. 3. To determine the role of protein kinase C (PKC) in the regulation of the choline co-transporter. 4. To determine the other second messenger system(s) directly involved in the regulation of the choline cotransporter. 5. To determine the specific ligands responsible for the direct activation of the second messenger systems involve in the relation of the choline co-transporter.